Fatty acid synthase (FAS), the enzyme that synthesizes the fatty acid palmitate, is overexpressed in prostate cancer and many other cancers of epithelial origin. We have discovered that orlistat, an FDA approved drug, is a novel inhibitor of the thioesterase (TE) domain of FAS. Inhibition of FAS by orlistat results in the selective killing of tumor cells in vitro and in vivo. The goal of this project is to determine the basic cellular and biochemical mechanisms of the anti-tumor effects of orlistat and other FAS inhibitors. To this end we have demonstrated that the endoplasmic reticulum (ER) stress response is activated in tumor cells upon FAS inhibitor treatment. Activation of the ER stress response appears to be upstream of apoptosis, perhaps engaging the cell death program. Moreover, the combination of FAS inhibitors with thapsigargin, another agent known to activate ER stress, yields a synergistic decrease in tumor cell survival. We have also determined the crystal structure of the TE domain (FAS-TE) in complex with orlistat. Orlistat binds to the active site in a manner contrary to a previously proposed model of substrate binding. Three specific aims are proposed to further explore these observations and to provide a critical foundation for the design and optimization of FAS inhibitors for cancer therapy. In Specific Aim 1 we will use tumor cell lines and transformed mouse embryonic fibroblasts (MEFs) with pathway specific mutations to determine the contribution of the PERK, IRE1 and ATF6 signaling pathways to the ER stress response when FAS is inhibited. In Specific Aim 2 the ability of thapsigargin and hypoxia to enhance the cytotoxic effects of FAS inhibitors will be determined. An in vivo imaging system will also be used to monitor ER stress-driven luciferase activity in tumor xenografts of mice treated with FAS inhibitors. In Specific Aim 3 we will use X-ray crystallography to determine the structural basis for the interactions between FAS-TE and orlistat and the orlistat analog Ebelactone B. We will also test whether substrate binds in a similar manner to orlistat by determining the crystal structures of substrate and product and analyzing the activity of site-directed mutants of conserved residues within the binding groove using a new, continuous assay. These studies will provide invaluable insights into how orlistat and other FAS inhibitors initiate apoptosis and how these compounds may be useful to increase the efficacy of current cancer drugs, particularly those where resistance has occurred. The proposed crystal structures will be instrumental to the rational design of analogs of orlistat with improved target specificity, bioavailability, and pharmacokinetics for the treatment of a variety of cancers.